Abstract
Phosphodiesterase type 2A (PDE2A) hydrolyzes cyclic nucleotides cAMP and cGMP, thus efficiently controlling cNMP-dependent signaling pathways. PDE2A is composed of an amino-terminal region, two regulatory GAF domains, and a catalytic domain. Cyclic nucleotide hydrolysis is known to be activated by cGMP binding to GAF-B; however, other mechanisms may operate to fine-tune local cyclic nucleotide levels. In a yeast two-hybrid screening we identified XAP2, a crucial component of the aryl hydrocarbon receptor (AhR) complex, as a major PDE2A-interacting protein. We mapped the XAP2 binding site to the GAF-B domain of PDE2A. PDE assays with purified proteins showed that XAP2 binding does not change the enzymatic activity of PDE2A. To analyze whether PDE2A could affect the function of XAP2, we studied nuclear translocation of AhR, i.e. the master transcription factor controlling the expression of multiple detoxification genes. Notably, regulation of AhR target gene expression is initiated by tetrachlorodibenzodioxin (TCDD) binding to AhR and by a poorly understood cAMP-dependent pathway followed by the translocation of AhR from the cytosol into the nucleus. Binding of PDE2A to XAP2 inhibited TCDD- and cAMP-induced nuclear translocation of AhR in Hepa1c1c7 hepatocytes. Furthermore, PDE2A attenuated TCDD-induced transcription in reporter gene assays. We conclude that XAP2 targets PDE2A to the AhR complex, thereby restricting AhR mobility, possibly by a local reduction of cAMP levels. Our results provide first insights into the elusive cAMP-dependent regulation of AhR.
Highlights
13656 JOURNAL OF BIOLOGICAL CHEMISTRY lyl and guanylyl cyclases PDEs regulate the amplitude and duration of cell signaling events mediated by cyclic nucleotides
We identify a functional interaction between Phosphodiesterase type 2A (PDE2A) and aryl hydrocarbon receptor (AhR) pathways that is mediated by XAP2, an essential component of the AhR complex, providing a direct link between cAMP- and TCDD-dependent pathways
Two of these glutathione S-transferase (GST) fusion proteins bound to PDE2A, and one of these was XAP2, a 330 amino acid protein of the immunophilin family (Fig. 1, A and B)
Summary
Materials—The full-length PDE2A cDNA (clone IRAKp961N0275Q2, GenBankTM accession number BC040974) was obtained from RZPD (Berlin, Germany). 8-Br-cAMP and dbcAMP were from Biolog (Bremen, Germany), and TCDD and forskolin were from Sigma. COS-1 cells transfected with expression constructs for VSV-PDE2A were lysed by scraping in 500 l of ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM MgCl2, including the following protease inhibitors: 1.12 mM dithiothreitol, 50 M chloroquine, 2 g/ml aprotinin, 1 g/ml leupeptin, and 1 g/ml phenylmethylsulfonyl fluoride). PDE2A Activity Assay—COS-1 cells (5 ϫ 10 cm dishes) transfected with pSG8-PDE2A or with empty pSG8 vector (mock) were harvested in 1 ml of lysis buffer and immunoprecipitated with 10 l of mouse anti-VSV antibody for 2 h followed by incubation with 200 l of protein A/G-PLUS agarose beads (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h. To verify expression of PDE, lysed cells were collected after each experiment, proteins were precipitated using acetone, solubilized in SDS sample buffer, and analyzed by SDS-PAGE and immunoblotting
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